Proline residue in L11 as a key regulator of translational GTPases?

The ribosome is run by translational GTPases. Translational GTPases, in their turn, are regulated by the ribosome. They all bind in the same region (GAC, GTPase associated center) of the ribosome. In bacteria the GTPase binding site consists of a couple of rRNA elements: SRL (sarcin-ricin loop) and thiostrepton loop and several ribosomal proteins:L7/L12 stalk (L10 and L7/L12) and L11.

The latter is the main hero of a fresh paper in Nature Structural and Molecular Biology by Wang and coworkers. They show that bacterial translational GTPases (such as EF-G) when binding to the ribosome act as peptidyl-prolyl cis-trans isomerases (PPIases) driving isomerisation in the conserved residue in the ribosomal protein L11. This isomerisation, in turn, transmits signal to the ribosomal protein L7 /L12 - something that is necessary for efficient GTPase cycling on the ribosome.

I like L11 - it is a key protein for stringent response, and without it stringent factor RelA does not work, as was shown using E. coli mutants lacking L11 (Dabbs J. Bac 1979). These mutants are perfectly viable, but grow ten times slower then the wild type E. coli, most probably due to defects in the ribosome assembly (Hampl et al. JBC 1981). The very viability of the L11 knock-out strains tells us that L11 is not the key for keeping the ribosome running. In fact, less than a half of ribosomal proteins can be knocked-out in E. coli (22 out of 54, Shoji et al. JMB 2011), making L11 one of the less-important ones... and keeping an eye of the translational GTPases is definitely not one of the less-important functions!

This seems to be bit paradoxical - a ribosomal protein that is dispensable involved in something that is very central for protein biosynthesis. It gets even more fascinating when you look at the evolutionary aspect of the story (Gem Atkinson does that in her blog). Wang and colleagues managed to map  the PPIase site of EF-G.  As they show PPIase activity is universal for all the bacterial translational GTPases they tested, and the PPIase site is, surprisingly, quite a variable region of the G domain! So, do they all reinvent the weel separately? This is all most peculiar.

References:

Wang et al. A conserved proline switch on the ribosome facilitates the recruitment and binding of trGTPases. Nat Struct Mol Biol (2012) PIMD: 22407015

Double life of mitochondrial ribosomal protein L7 12

Mitochondria have their own transcriptional and translational apparatus, even though they produce only a handful of proteins, therefore most of the proteins are imported from the cytoplasm. Trancription, translation and protein insertion into the membrane are interconnected: translational activators regulating mitochondrial translation are interacting with mitochondrial RNA polymerase via Nam1p and Sls1p proteins (Bryan et al. Genetics 2002), Puf proteins connect cytoplasmic translation and protein import into mitochondria by direct interaction with Tom20 subunit of the TOM protein import channel (Saint-Georges et al. PLoS ONE 2008).

But this seems not tight enough interaction for mitochondrial translation and transcription. It turnes out what mitohondrial ribosomal protein L7 12 (the one that brings translational GTPases to the ribosome), has a double life. Apart from doing its normal job as a part of the ribosome, it doubles as a transctiptional factor, selectively associating with human mitochondrial RNA polymerase and activating it (Surovtseva et al. PNAS 2011). And as if it is not enough, there are several paralogues of L7 12 in mitochondria, both in plants (Delage et al. Biochimie 2007) and in mammals (Koc et al. JBC 2001). 

Observing protein synthesis inside the living mammalian cell

Biologists really love seeing things for themselves. Take for instance the central dogma: DNA - RNA - protein. It all well and good when represented as childish-looking blobs fooling around and passing amino acids one to each other, but how about it actually happening in the real 3D cell stuffed with other goodies? Well, obviously, people tried looking into the question.

The first option is you can to label fluorescently some components of the machinery - ribosomes, mRNAs, factors - and plonk the bug under the microscope. The problem here is that you can't really see what these components are doing - are they idle? are they active? are they so fucked up by adding the fluoresecnt label that now they are being rapidly degraded? One possible solution is to interfere with the cell inhibiting some crucial step the component of interest is involved in and see what happens to the distribution of the labeled component. For instance, one can add an antibiotic. The problem with this approach is that you a) interfere with the system b) interfere with the system c) antibiotics are often fluorescent, so you interfere with the system even more.

One solution that is often used in vitro for converting fluorescence signal into the distance signal is by using FRET, Förster resonance energy transfer (for an excellent review of FRET-based single molecule investigations of translation in vitro see Blanchard). The idea behind FRET is that one uses two fluorofores at the same time. Fluorofore 1 is excited at wavelength λ1, and emits at λ2. Fluorofore 2 is adsorbs at λ2 and emits at λ3. In order for all this cascade to work, the two fluorofores have to be very, very close because efficiency of FRET decreases with distance with an inverse 6th power law.

Now, back to observing translation in vivo. Barhoom at al. exploited the FRET strategy in their piece that just came out in NAR. They used a FRET pair consisting of two labeled tRNAs. These two are getting very close on the ribosome, generating a FRET signal (a strategy recently used in vitro by Uemura at al.). And they are getting close on the ribosome only if they are actively engaged in translation (Fig. 1). Therefore by looking at the tRNA FRET Barhoom and colleagues can observe spots of active translation inside the cell under different conditions, such as viral infection etc.

The paper is open access, so do take advantage of that.

Fig. 1. Two labeled tRNAs are constituting a FRET pair. When they are in close proximity on the ribosome they produce a FRET signal. When floating about in the cytoplasm, they are too far to generate FRET.


PS: this post is part of the MolBio carnival Nr 14!

References:

Blanchard SC (2009). Single-molecule observations of ribosome function. Current opinion in structural biology, 19 (1), 103-9 PMID: 19223173

Barhoom S, Kaur J, Cooperman BS, Smorodinsky NI, Smilansky Z, Ehrlich M, & Elroy-Stein O (2011). Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA. Nucleic acids research PMID: 21795382

Uemura S, Aitken CE, Korlach J, Flusberg BA, Turner SW, & Puglisi JD (2010). Real-time tRNA transit on single translating ribosomes at codon resolution. Nature, 464 (7291), 1012-7 PMID: 20393556

Adjacent gene pairing and regulation of protein expression

Just had a great talk by Mike McAlear who was visiting us on his way to Poland. He gave a talk about co-regulation of adjustment genes, specifically ribosomal proteins and ribosomal assembly enzymes. I know what you are thinking about now, transcriptional read-through and the like. But it seems to be much more fun than that!

Genes can be on the opposite strands, they can face in opposite directions, but still, there is co-regulation which can not be contributed simply to a shared regulatory system. Something with chromatin structure, probably. Being positioned next to each other was shown to be an important factor for noise in protein expression. Becsei at al. showed in yeast that noise in protein expression is sensitive to gene position in the chromosome, and, consequently, genes positioned next to each other show somewhat correlated behavior. I think it is all different sides of one story...

References:

Adjacent gene pairing plays a role in the coordinated expression of ribosome biogenesis genes MPP10 and YJR003C in Saccharomyces cerevisiae. Arnone JT, McAlear MA. Eukaryot Cell. 2011 Jan;10(1):43-53. PIMD: 21115740

Prime movers of noisy gene expression. Paulsson J. Nat Genet. 2005 Sep;37(9):925-6. PIMD: 16132049

Contributions of low molecule number and chromosomal positioning to stochastic gene expression. Becskei A, Kaufmann BB, van Oudenaarden A. Nat Genet. 2005 Sep;37(9):937-44. PIMD: 16086016

Co-expression of adjacent genes in yeast cannot be simply attributed to shared regulatory system. Tsai HK, Su CP, Lu MY, Shih CH, Wang D. BMC Genomics. 2007 Oct 3;8:352. PIMD: 17910772

Single-molecule investigations of the stringent response machinery in living bacterial cells

Wikipedia: "reductionism, an approach to understanding the nature of complex things by reducing them to the interactions of their parts, or to simpler or more fundamental things". This approach was very successful in unrevealing the basic mechanisms of biological systems. Modern biochemistry is reductionism in its pure form: we purify individual components, mix them together in a test tube and make this in vitro system jump through the hoops and this way we learn how it works. Then we extrapolate what we learned from the in vitro system to the cell, and test our model in vivo: overexpress some components, knock-out the other, introduce mutations etc.

However, sometimes producing in vitro system is not feasible, either because it is to laborious or because we simply do not know what are the components. A good solution would be then to do biochemistry, but... inside the living cell. This approach became technically feasible in the recent decades, and was highly successful in cracking these hard problems for which in vitro investigations are just not cutting it. In vivo biochemistry relies on labeling the protein (proteins) of interest with a fluorescent tag, usually a GFP derivative, and then following its movement inside the living cell on the single molecule level. Movement of the protein can tell us about its functional cycle: binding to a partner will slow its diffusion, for instance.

Now this approach was applied to investigation of the stringent response (I have discussed this fascinating bacterial adaptation system quite at length here). In short, when bacteria are starving for amino acids, they accumulate deacylated tRNAs. These bind to the ribosomal A-site, and this situation is sensed by a protein called RelA, which starts producing alarmone molecule ppGpp. One important thing about RelA functional cycle is that it has two states with distinctly different difusion properties: ribosome bound and free.

This was taken advantage of in the recent paper by English at al. RelA was labelled with a fluorescent GFP variant and its diffusion was followed at ms time resolution. Indeed, inactive RelA turned out to be tightly associated with the ribosomes and diffusing slowly (Fig. 1). However, when stress was induced, either by amino acid limitation or by the heat shock, RelA fell off the ribosome and started moving about much, much faster (Fig. 1).

It is known that under these conditions RelA is enzymatically active and produces ppGpp. Since active RelA seems to spend its time off, rather than on the ribosome, it was suggested that ppGpp production is happening off the ribosome as well. And this is a rather unique mechanism for a ribosome-associated factor. Usually on the ribosome is when the protein is active: RelE binds to the ribosome and cuts the mRNA, EF-G binds, hydrolyses GTP and translocates A and P site tRNAs, ricin binds and cuts the ribosomal RNA.

Fig. 1. MSD (Mean Square Displacement) analysis of the RelA diffusion in vivo. Diffusive behavior of active and inactive RelA is compared to that of ribosomes carrying fluorescent label on L25 protein (green triangles) and freely diffusing protein mEos2. Insert shows the difference in the individual trajectories of active (right trajectory) and inactive (left trajectory) RelA.

Now, of course, this mechanism of RelA has to be tested by other methods. As any approach, single molecule tracking in its current form has its limitations, and the biggest one is the labels used, GFP in this case. RelA fused with GFP is not RelA, it can behave somewhat different.

PS: and now this story was covered in the news! HFSP and UppsalaBio (in Swedish). Also it is covered as a Research highlight in Biopolymers.

PPS: a great review of the single molecule investigations in vivo just came out in Nature: Gene-Wei Li and Sunney Xie (2011). Central dogma at the single-molecule level in living cells. Nature, 475, 308-315 PIMD 21776976. Too bad, we are not mentioned!

PPPS: this blog post is covered in The MolBio Carnival #13!

PPPPS: and now our paper made it to F1000.

References:

Xie XS, Choi PJ, Li GW, Lee NK, & Lia G (2008). Single-molecule approach to molecular biology in living bacterial cells. Annual review of biophysics, 37, 417-44 PMID: 18573089

Potrykus K, & Cashel M (2008). (p)ppGpp: still magical? Annual review of microbiology, 62, 35-51 PMID: 18454629

Gallant J, Palmer L, & Pao CC (1977). Anomalous synthesis of ppGpp in growing cells. Cell, 11 (1), 181-5 PMID: 326415

Brian P. English, Vasili Hauryliuk, Arash Sanamrad, Stoyan Tankov, Nynke H. Dekker, and Johan Elf (2011). Single-molecule investigations of the stringent response machinery in living bacterial cells PNAS 108(31), E359-364 PIMD: 21730169 and the PNAS Author Summary

Mendeley group on stringent response

Antibiotics vs the ribosome

Ok, now it is official: this summer in Tartu there will be a conference on antibiotics inhibiting protein synthesis organized by Tanel Tenson.

It is not too late to register! And it has a very, very competitive registration fee - 0 USD!

Confirmed speakers:

James Williamson (Scripps Research Institute),
Alexander Mankin (University of Illinois at Chicago),
Steven Douthwaite (University of South Denmark),
Daniel Wilson (University of Munich),
Karen Shaw (Trius Therapeutics),
Ada Yonath (Weizmann Institute of Science).
Birte Vester (University of Southern Denmark)
Joyce Sutcliffe (Tetraphase Pharmaceuticals)
Mans Ehrenberg (Uppsala University)
Chaitan Khosla (Stanford University)
Markus Zeitlinger (Medical University of Vienna)

... and me and Gem Atkinson! That sold it, right?

Antibiotics affecting ribosome's protein composition

Antibiotics kill bugs; and about a half of them are doing so by messing up translation. That usually means that the ribosome is stalled at a certain step, be it initiation or elongation or ribosomal recycling.

But it is not always just that. Sometimes antibiotics also mess up the ribosome itself  and affect its composition.

Exhibit A: kasugamycin, an antibiotic that inhibits translation initiation in bacteria by interfering with binding of the the initiator tRNA. Amazingly enough, treatment with kasugamycin results in dramatic change in the ribosomal composition which is in turn changing ribosome's functional properties. Several proteins dissociate from the small ribosomal subunit (S1, S2, S6, S12, S18 and S21) which turns the 70S ribosome into a 61S kasugamycin particle. Ribosomal protein S1 is of particular interest here, because it is very important for the mRNA:ribosome interactions and is responsible for  A/U rich sequences acting as translational activators.

The S61 particle loses the ability to translate mRNAs with Shine-Dalgarno sequences, while being able to translate leaderless mRNAs, that is the ones starting directly with the initiation codon at the 5'. These leaderless mRNAs can be translated without the help of any initiation factors, by bacterial and eukaryotic ribosomes alike, so it is no surprise that S61 particles, even though compromised can still translate these messages.

What is particularly interesting in the kasugamycin story, it is that loss of the ribosomal proteins can be reconstituted in vitro by simply mixing the drug with the 70S. This means that the effect is direct rather than mediated by the assembly process (see below for an example of the latter effect).

Exhibit B: chloramphenicol and erythromycin. These antibiotics cause defects of the ribosomal assembly, and they seem to be doing so by interfering with the expresion levels of different ribosomal proteins. Here we have Liebig's barrel in action: you interfere with levels of many components you need to have and end up running out of one, the limiting one.

All of the above is highly relevant for people using antibiotics as tools, for instance in microscopy. Chloramphenicol and kasugamycin are widely used to inhibit translation (for instance here and here). It's worth remembering that they are doing much more than that while interpreting your results. Sometimes the tool you use can have much more complicated character than one would anticipate, as I discussed here.

References:

Wilson DN (2009). The A-Z of bacterial translation inhibitors. Critical reviews in biochemistry and molecular biology, 44 (6), 393-433 PMID: 19929179

Schluenzen F, Takemoto C, Wilson DN, Kaminishi T, Harms JM, Hanawa-Suetsugu K, Szaflarski W, Kawazoe M, Shirouzu M, Nierhaus KH, Yokoyama S, & Fucini P (2006). The antibiotic kasugamycin mimics mRNA nucleotides to destabilize tRNA binding and inhibit canonical translation initiation. Nature structural & molecular biology, 13 (10), 871-8 PMID: 16998488

Schuwirth BS, Day JM, Hau CW, Janssen GR, Dahlberg AE, Cate JH, & Vila-Sanjurjo A (2006). Structural analysis of kasugamycin inhibition of translation. Nature structural & molecular biology, 13 (10), 879-86 PMID: 16998486

Kaberdina AC, Szaflarski W, Nierhaus KH, & Moll I (2009). An unexpected type of ribosomes induced by kasugamycin: a look into ancestral times of protein synthesis? Molecular cell, 33 (2), 227-36 PMID: 19187763

Siibak T, Peil L, Dönhöfer A, Tats A, Remm M, Wilson DN, Tenson T, & Remme J (2011). Antibiotic-induced ribosomal assembly defects result from changes in the synthesis of ribosomal proteins. Molecular microbiology, 80 (1), 54-67 PMID: 21320180

Nevo-Dinur K, Nussbaum-Shochat A, Ben-Yehuda S, & Amster-Choder O (2011). Translation-independent localization of mRNA in E. coli. Science (New York, N.Y.), 331 (6020), 1081-4 PMID: 21350180

Tzareva NV, Makhno VI, & Boni IV (1994). Ribosome-messenger recognition in the absence of the Shine-Dalgarno interactions. FEBS letters, 337 (2), 189-94 PMID: 8287975

Andreev DE, Terenin IM, Dunaevsky YE, Dmitriev SE, & Shatsky IN (2006). A leaderless mRNA can bind to mammalian 80S ribosomes and direct polypeptide synthesis in the absence of translation initiation factors. Molecular and cellular biology, 26 (8), 3164-9 PMID: 16581790

Montero Llopis P, Jackson AF, Sliusarenko O, Surovtsev I, Heinritz J, Emonet T, & Jacobs-Wagner C (2010). Spatial organization of the flow of genetic information in bacteria. Nature, 466 (7302), 77-81 PMID: 20562858

Abort! Abort!

Sometimes things go so wrong that it is just easier to start all over again. Bacteria have these situations too - it's not just us, humans! - and the central dogma of molecular biology (DNA replication, transcription and translation) is no exception.

In essence all the three steps of the central dogma share the very same basic topology: there is a message that gets read, there is a tool that reads it and there is a product. It looks like so:

Say, in the case of translation mRNA (the message) gets read by the ribosome (the tool) and protein (the product) is produced. And when things go wrong, there are three things you can abort: the message, the product and the tool. Let us see how it goes.

Replication

DNA polymerase (the tool) reads the DNA (the message) and produces DNA (the product). And when wrong nucleotide is incorporated, DNA polymerase can excise it and continue making the product using so called  proof-reading mechanism. Complete abortion of the growing DNA strand does not happen, and if mistake is done, it is done and you live with it. Surely, there are ways to fix it later (recombination and so on), but not on the spot, during the replication.

Transcription


RNA polymerases can proof-read too. However, many more things can be done. Special set of transcription factors, called GreA and GreB in bacteria and TFSII in eucaryotes, can activate intrinsic hydrolytic activity of the RNA polymerase and cleave off the growing product. Stalled complex is resolved and now we can try again.

Translation


First, there is a proof-reading mechanism, but rather than cutting off the mis-incorporated letter, GTP is hydrolyzed by GTPase EF-Tu which brings the aminoacyl-tRNA.

Second, if the mistake is done, and wrong amino acid was incorporated after all, bacterial class-1 release factors RF1 and RF2 become prone to peptide-release independent of the stop codon, thus removing the product (the growing protein chain). In mitochondria translational system is bacterial-like, but much more insane, and several (as many as 4 in humans!) class-1 release factors are present, with some of them lacking the ability to recognize the stop codon at all (ICT1, for example), and these resolve stalled ribosomal complexes by cutting off the peptide as well as their bacterial counterparts.

Third, bacterial toxins such RelE and the like are resolving ribosomal complexes by cutting the message (mRNA) rather than the product. Calling them toxins is rather misguiding, they are more of the rescue factors.

And lastly, eukaryotic translational factors Dom34 and Hbs1 (related to termination factors eRF1 and eRF3) are splitting the stalled ribosome into subunits, re-setting the tool.

So it seems the further we move from the DNA, the more dispensable the production complex becomes: in the case of DNA polymerases we have only proof-reading, RNA polymerases can do that and also cleave the message, and translational machinery can do it all: cutting the message (RelE), cutting the product (release factors) and resetting the tool by splitting the ribosome into subunits (Dom34 and Hbs).

References:

Borukhov S, Sagitov V, & Goldfarb A (1993). Transcript cleavage factors from E. coli. Cell, 72 (3), 459-66 PMID: 8431948

Toulmé F, Mosrin-Huaman C, Sparkowski J, Das A, Leng M, & Rahmouni AR (2000). GreA and GreB proteins revive backtracked RNA polymerase in vivo by promoting transcript trimming. The EMBO journal, 19 (24), 6853-9 PMID: 11118220

Pedersen K, Zavialov AV, Pavlov MY, Elf J, Gerdes K, & Ehrenberg M (2003). The bacterial toxin RelE displays codon-specific cleavage of mRNAs in the ribosomal A site. Cell, 112 (1), 131-40 PMID: 12526800

Orlova M, Newlands J, Das A, Goldfarb A, & Borukhov S (1995). Intrinsic transcript cleavage activity of RNA polymerase. Proceedings of the National Academy of Sciences of the United States of America, 92 (10), 4596-600 PMID: 7538676

Kassavetis GA, & Geiduschek EP (1993). RNA polymerase marching backward. Science (New York, N.Y.), 259 (5097), 944-5 PMID: 7679800

Richter R, Rorbach J, Pajak A, Smith PM, Wessels HJ, Huynen MA, Smeitink JA, Lightowlers RN, & Chrzanowska-Lightowlers ZM (2010). A functional peptidyl-tRNA hydrolase, ICT1, has been recruited into the human mitochondrial ribosome. The EMBO journal, 29 (6), 1116-25 PMID: 20186120

Shoemaker CJ, Eyler DE, & Green R (2010). Dom34:Hbs1 promotes subunit dissociation and peptidyl-tRNA drop-off to initiate no-go decay. Science (New York, N.Y.), 330 (6002), 369-72 PMID: 20947765

Atkinson GC, Baldauf SL, & Hauryliuk V (2008). Evolution of nonstop, no-go and nonsense-mediated mRNA decay and their termination factor-derived components. BMC evolutionary biology, 8 PMID: 18947425

Antonicka H, Ostergaard E, Sasarman F, Weraarpachai W, Wibrand F, Pedersen AM, Rodenburg RJ, van der Knaap MS, Smeitink JA, Chrzanowska-Lightowlers ZM, & Shoubridge EA (2010). Mutations in C12orf65 in patients with encephalomyopathy and a mitochondrial translation defect. American journal of human genetics, 87 (1), 115-22 PMID: 20598281

Zaher HS, & Green R (2009). Quality control by the ribosome following peptide bond formation. Nature, 457 (7226), 161-6 PMID: 19092806

Observer effect in biology: Schrödinger's cat mitochondria

All quantum physicists know that observation itself changes the object of observation. We will never know what things are actually doing when we are not looking, just because if in order to figure out what they do, we need to look; it's catch-22. But that's quantum physics, you say. How about molecular biology?

Well, here is an example. Mitochondria, as you know, have their own genome, and they translate it, and they do so in a very funky way. Ever translation termination is peculiar. It is a variation of bacterial translation termination, but different. There are two mitochondrial class-1 release factors (the ones which actually recognize the stop codon and cleave off the peptide): mtRF1a and mtRF1. mtRF1a is an omnipotent release factor and it recognizes normal stop codons UAA and UAG, as it was proved biochemically in vitro. mtRF1... this one is a bit tricky.

First idea is that it recognizes funky stop codons like AGA and AGG (together - AGA/G), which are indeed present in mitochondria. Biochemistry in heterologous system seems to support this one.

Second is that there is no need for mtRF1 at all, and AGA/G stop codons actually never get read at all, therefore there is no need to recognize these! Wow, that's radical and this is why it is published in Science. This story is the subject of this post.

So... how did they figure it out. They check for ribosomal positioning on the termination codon and they figure out that it seems to slip (frame-shift) from the non-standart uAGA/G codon backward and ends up with classical UGAa/g in the A-site. Bang, problem solved, we do not need to recognize the strange stop codon and thus there is no need for mtRF1 at all. Clever. But how do they see it?

They use bacterial toxin RelE. This peculiar molecule binds in the ribosomal A-site and cleaves mRNA there. It works in bacteria, eucaryotes and, obviously, mitochondria because the ribosome is so darn conserved. However, RelE does not cleave all the codons with the same efficiency, it has very strong preferences for certain sequences - such as regular stop codons, UGA or UGG!

Fig. 1 RelE efficiency is different for different codons, lifted from Pedersen at al. 2003

Looking at the x-ray structure of RelE in the complex with mRNA and 70S ribosome we can see why: it is all down to the interactions between the specific residues in RelE and mRNA. If these residues are not there, there will be no interaction and no cleavage - see Fig. 2.

Fig. 2 Proposed reaction mechanism for RelE-mediated cleavage, lifted from Neubauer at al., 2009.

And now - back to the Schrödinger's cat. When researchers used RelE to probe for position of the mitochondrial ribosome on the mRNA, all the cleavages detected were with UAG in the A-site. Why? Well, because this is where RelE can cut, so it cleaved there. It may have even caused this frame-shift. Why didn't they see any ribosomes on the AGG? well, because RelA does not want to cleave there!

So... may be the tool used for observation changed the system and told us something about itself (something that we already knew). Not about the system! Still, it's a Science paper, hey. And the idea is very, very cute!

And, of course, I can be completely wrong!

Fig. 3 Schrödinger's cat. Not really related to RelE at all.


PS: as it turnes out, the problem of affecting the biological system while studying it was discussed by at length here: Bridson EY, & Gould GW, Quantal microbiology.


References:

Neubauer C, Gao YG, Andersen KR, Dunham CM, Kelley AC, Hentschel J, Gerdes K, Ramakrishnan V, & Brodersen DE (2009). The structural basis for mRNA recognition and cleavage by the ribosome-dependent endonuclease RelE. Cell, 139 (6), 1084-95 PMID: 20005802

Andreev D, Hauryliuk V, Terenin I, Dmitriev S, Ehrenberg M, & Shatsky I (2008). The bacterial toxin RelE induces specific mRNA cleavage in the A site of the eukaryote ribosome. RNA (New York, N.Y.), 14 (2), 233-9 PMID: 18083838

Pedersen K, Zavialov AV, Pavlov MY, Elf J, Gerdes K, & Ehrenberg M (2003). The bacterial toxin RelE displays codon-specific cleavage of mRNAs in the ribosomal A site. Cell, 112 (1), 131-40 PMID: 12526800

Young DJ, Edgar CD, Murphy J, Fredebohm J, Poole ES, & Tate WP (2010). Bioinformatic, structural, and functional analyses support release factor-like MTRF1 as a protein able to decode nonstandard stop codons beginning with adenine in vertebrate mitochondria. RNA (New York, N.Y.), 16 (6), 1146-55 PMID: 20421313

Soleimanpour-Lichaei HR, Kühl I, Gaisne M, Passos JF, Wydro M, Rorbach J, Temperley R, Bonnefoy N, Tate W, Lightowlers R, & Chrzanowska-Lightowlers Z (2007). mtRF1a is a human mitochondrial translation release factor decoding the major termination codons UAA and UAG. Molecular cell, 27 (5), 745-57 PMID: 17803939

Temperley R, Richter R, Dennerlein S, Lightowlers RN, & Chrzanowska-Lightowlers ZM (2010). Hungry codons promote frameshifting in human mitochondrial ribosomes. Science (New York, N.Y.), 327 (5963) PMID: 20075246

Lekomtsev SA (2007). Non-standard genetic codes and translation termination. Molekuliarnaia biologiia, 41 (6), 964-72 PMID: 18318113

Bridson EY, & Gould GW (2000). Quantal microbiology. Letters in applied microbiology, 30 (2), 95-8 PMID: 10736007

All ribosomes are equal, but some ribosomes are more equal than others

There are many ways of regulating translation - different mRNA structures, modifications of the canonical set of translation factors, specialized factors and so on. Well, you can also have different ribosomes and they may have different functions.

Here is a review about different ribosomal flavors. Main points:

1. rRNA can be modified differently under different conditions, thus resulting in ribosomes with different properties, such as thermal stability, affinity between 30S and 50S, etc. Here it an example of ITC used for studying these rRNA-modified ribosomes.

2. Ribosomes can have different rRNA and proteins when produced under different conditions. The most striking example is Haloarcula marismortui with 3 rRNA operones, out of which one codes extremely divergent copy which is expressed at high temperatures. You mess with it and bug becomes temperature sensitive.

3. Profs of functional differences of these ribosomes - here we do not have much. Stability - yes, see above. But function...

Well, we do have a bunch of proteomics data showing that in Saccharomyces cerevisiae different paralogues of r-proteins localize differently and are specifically involved in translation of some mRNAs.

A quick reminder - Saccharomyces cerevisiae had a whole genome duplication (WGD), thus they generally have loads of paralogues and thus are used to study evolution of proteins after duplication. Apart from yeast, WGD has happend in many other lineages (bony fish, plants), and it would be interesting to see what happens to r-proteins during WGD...

Back to the functionality of different ribosomes. One interesting possible functional regulation is discussed. Knocking out non-essential ribosomal protein Rps25 makes ribosomes incapable of translating some IRESes, though no effect on normal cap-dependent translation. Is expression of Rps25 regulated during viral invasion? No evidence of that as yet.

4. There are two possible ways of using different ribosomes:

First it can be that when the cell changes its ribosomal set, changes one flavor for another, no mixing - a global rewiring of the translational machinery. This seems to be the case for rRNA modification in bacteria - appropriate enzyme is induced  under certain conditions and all the ribosomes are modified, viola. Same for different rRNA genes in archaea.

Alternative approach is to have many different ribosomes for different mRNAs. This is seemingly what we have in yeast (see above). Specific localization of different ribosomes and use of different mRNA-specific factors would then ensure proper coupling of appropriate ribosome with the right mRNA. Different localization of different paralogues of r-proteins in Saccharomyces cerevisiae is shown experimentally, and these proteins have different requirements for assembly into the 80S.

PS: what all the ribosomes have in common is their color. They all are yellow.

References:

1. Gilbert VW. Functional specialization of ribosomes? (2011) Trends. Biochem. Sci. 2011 PIMD 21242088

2. Lopez-Lopez at al. Intragenomic 16S rDNA divergence in Haloarcula marismortui is an adaptation to different temperatures. (2007) J. Mol. Evol. 65, 687–696

3. Esguerra J. et al. Functional importance of individual rRNA 20-O-ribose methylations revealed by high-resolution phenotyping. (2008) RNA 14, 649–656

4. Komilli at al. Functional specificity among ribosomal proteins regulates gene expression. (2007) Cell PIMD 17981122

4. Kellis at al. Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisiae (2007) Nature v. 131 pp 557-571

Z-RNA–binding domain Zα as ribosomal inhibitor: fishing for ribosomes

Feng at al. in NSMB show that Z-RNA (or DNA) binding domain Zα inhibits ribosomal function. Binds to the ribosome and inhibits it! Basically does what ribosome-binding antibiotics do - they bind, freeze the ribosome in some particular conformation and thus inhibit it. Viomycin can be a gerat example of that.

Better still, Zα seems to bind ribosomes nondiscriminantly (both bacterial and mammalian), so using a column with immobilized Zα you could purify ribosomes from whatever cells you have. Sounds fun, and, as I learned from the PI behind the paper - they are working on it, though this practical application is not mentioned in the original paper.

References:

Feng S, Li H, Zhao J, Pervushin K, Lowenhaupt K, Schwartz TU, & Dröge P (2011). Alternate rRNA secondary structures as regulators of translation. Nature structural & molecular biology PMID: 21217697

Ermolenko DN, Spiegel PC, Majumdar ZK, Hickerson RP, Clegg RM, & Noller HF (2007). The antibiotic viomycin traps the ribosome in an intermediate state of translocation. Nature structural & molecular biology, 14 (6), 493-7 PMID: 17515906

Using isothermal titration calorimetry for following subunit association

We use Isothermal Titration Calorimetry (ITC) for studying translational GTPases for quite some time now, and it turnes out one can do one more translation-related things with it: measure thermodynamics of subunit association with it (Osterman Biochimie 2011).

Quite amazing, actually. Sample is mixed at 200 rpm, ∼2 μM 50S are titrated with ∼11 μM 30S subunits, and yet seemingly there are no problems with precipitation and heat generated by it.

I would suspect that this means that one can follow initiation complex formation too...

References:

Ilya A Osterman, Petr V Sergiev, Philipp O Tsvetkov, Alexander A Makarov, Alexey A Bogdanov, Olga A Dontsova. Methylated 23S rRNA nucleotide m(2)G1835 of Escherichia coli ribosome facilitates subunit association. Biochimie 2011 PIMD 21237242

Ribosome-assisted protein UN-folding

"Stand still, do not move! I gave you life, I will also kill you!" said Taras, and, retreating a step backwards, he brought his gun up to his shoulder. Andrii was white as a sheet; his lips moved gently, and he uttered a name; but it was not the name of his native land, nor of his mother, nor his brother; it was the name of the beautiful Pole. Taras fired.

Taras Bulba, Nikolai Vasilievich Gogol

Ribosome makes proteins, we all know that. But producing a string of amino acids is just a half it. In order to be functional, nascent protein should fold correctly. And the ribosome takes care of that as well.

First, building blocks of the protein - alfa helices, for instance - are formed already in the ribosomal tunnel where they are protected from the hostile environment. When the protein emerges outside of the tunnel, it is greeted by the ribosome-associated chaperons, which help it to fold. Moreover, by cleverly fine-tuning translational rate, protein syntheses machinery allows protein domains to be produced one by one with pauses in between, ensuring that they fold correctly.

And here is a surprise. A fresh paper in JACS by O'Brian and colleagues suggest that the ribosome destabilizes unfinished proteins dangling out of the ribosome tunnel. Coarse-grain simulations allow dissecting the nature of this phenomenon.

Stability of the protein is reflected by the Gibbs free energy of folding (ΔG). Gibbs free energy in turn can be divided into and enthalpic (ΔH) and entropic components (TΔS), ΔG = ΔH - TΔS. When the protein is close to the ribosome, it pays for it in freedom (obviously, it can't move about freely any more), which means that the total number of available microstates is lower. And S is dependent on the total number of these microstates, thus it goes down, bringing down the Gibbs free energy. 

Moreover, it is not just thermodynamics, it is kinetics too: ribosome decreases folding rate, and unfolding rate increases. This is keeping with the basic definitions relating the equilibrium constant (K) with the rate ones (k₊₁,  k_-1), ΔG = - RTln(K), K = k₊₁ / k_-1.

So why is the ribosome so nasty to its progeny? Well, I guess the answer is - it just can't help it. The effects described above are simple consequences of the fact that nascent protein has to be attached to the ribosome during translation, and this results in loss in available microstates and so on (see above). And translational machinery is trying hard to be kinder to nascent proteins, cleverly designing the ribosomal tunnel, employing chaperones and waiting for the domains to fold before moving to the next one.

But is the ribosome doing anything good to the folding protein? And yes, it does. It stirs the folding pathway towards more native-like intermediates, guiding the direction of folding so that it starts from the N-terminus - the part that gets translated first. And again, this beneficial effect is done again by restricting proteins' freedom - freedom to sample configurations diverging from the productive folding path. 

Isn't it a great example of excellent parenting?  

References:

O'Brien EP, Christodoulou J, Vendruscolo M, & Dobson CM (2011). New Scenarios of Protein Folding Can Occur on the Ribosome. Journal of the American Chemical Society PMID: 21204555

You pay in ribosomes for proteins

Everything costs. When cell grows, it needs energy and in needs materials. By the end of the day it comes down to accounting: if you need to make N proteins, you will need X ATPs molecules, Y aminoacids and Z ribosomes to do the job. And of all these ribosomes are the most expensive to make: they are huge, made of RNA and if you want to make proteins fast, you need lots of ribosomes!

So research team led by famous systems biologist Uri Alon decided to quantify the cost of making a protein. In order to do so, they forced E. coli producing Green Fluorescent Protein (GFP) which is inert and easy to quantify. They figured out that early in the exponential phase the cost of GFP (that is decrease in growth rate associated with production of a given amount of GFP) is high, but later on it markedly recreases!

This observation makes immediate sense: first you need to produce the tools for producing GFP (ribosomes, energy in the form of ATP etc.) and if instead of this you make GFP this GFP comes at a high prise. Corroborating with this logic, they figured out that if you transfer bacteria from energy-reach media to energy-poor, the price for GFP is low: well, you accumulated all these ribosomes during the good times, so now you can make some GFP cheap.

There could be an interesting connection here with another resent paper where in yeast it was shown that the cost of GFP is dramatically different for stable and denaturation-prone variants (for brilliant discussion of this paper see this post in It Takes 30). Is GFP equally stable in E. coli during the early and late exponential phase? Could it be that the effects observed here are reflecting mere change in GFP stability? Intracellular conditions do change in E. coli under different conditions, so it is possible that GFP is not always equally stable, and this may affect its physiological cost. Surprisingly, another report claims that in E. coli aggregated and soluble LacZ have very similar cost, which to some extent dispels my worries about GFP stability and cost.

The read-out for GFP quantification could be affected by cellular milieu as well: judging from my experience, GFP is definitely not always equally bright, and this could affect estimates of GFP concentration and thus the estimates of its physiological cost.

Discovering differences in the GFP cost in early and late exponential phase prompted the authors to try figuring out what cellular system is behind it. And they had a very good initial guess. In bacteria adaptation to changes in availability of food are regulated by the stringent response mechanisms, with RelA and SpoT proteins doing the job (see my previous posts on that subject, see 1 and 2). RelA produces ppGpp molecule that compels the cell to stop producing ribosomes and concentrate on aminoacid production, and SpoT mostly degrades ppGpp: too much of it would lead to complete inhibition of ribosomal production and eventually - death.

Therefore it is only logical that in the paper in question different knock-out strains missing RelA and SpoT were tested, and indeed, stringent response machinery turned out to the the key to radical changes of the protein cost during different stages of bacterial growth.

And here is the catch.

One of the strains they used was SpoT(-) RelA(+) strain, that is one having NO SpoT and allegedly INTACT RelA. As we discussed above this bug should be very much dead, and as yet no one managed to produce this strain. So what's about the strain presented in the paper then?

Well, there are many options. When you want, really want to knock out a gene, you finally succeed. However, bacteria want to live, and you select the ones with mutations that compensate for the knock-out of the gene you have. For instance, you can mutate main target of ppGpp, the RNA polymerase and make it insensitive to regulation. Aslo, you can mutate RelA and make it inactive. And there are several other possible compensatory mutations... In order to notice these changes in the strain you made you really need to run a lot of tests, and the authors did not.

So here is another example how bacteria are cleverly trying to fool systems biology approach (another example is here).

Update: the SpoT knock-out strain used in the original paper indeed was iffy, it had compensatory mutations in RelA and an erratum was published, which I discuss here.

References:

Shachrai I, Zaslaver A, Alon U, & Dekel E (2010). Cost of unneeded proteins in E. coli is reduced after several generations in exponential growth. Molecular cell, 38 (5), 758-67 PMID: 20434381

Potrykus K, & Cashel M (2008). (p)ppGpp: still magical? Annual review of microbiology, 62, 35-51 PMID: 18454629

Geiler-Samerotte KA, Dion MF, Budnik BA, Wang SM, Hartl DL, & Drummond DA (2010). Misfolded proteins impose a dosage-dependent fitness cost and trigger a cytosolic unfolded protein response in yeast. Proceedings of the National Academy of Sciences of the United States of America PMID: 21187411

Plata G, Gottesman ME, & Vitkup D (2010). The rate of the molecular clock and the cost of gratuitous protein synthesis. Genome biology, 11 (9) PMID: 20920270

Mendeley group on stringent response